Alzheimer's disease (AD) is an idiopathic progressive dementia that will affect a large percentage of our aging population. To date its etiology is still unknown. The neurodegenerative disease is characterized by a chronically deteriorating course of impaired intellectual function and memory loss.
Relatively little is known of the pathophysiological chain of events that leads to the premature dysfunction and death of affected neurons in Alzheimer's disease patients. Multiple abnormalities have been reported in the brains of patients who have been diagnosed as having Alzheimer's disease, but it is difficult to determine which of these are the result of brain damage and which contribute to premature neuronal dysfunction and death.
At the present time, the clinical diagnosis of Alzheimer's disease is one of exclusion. Secondary causes of loss of memory and impaired cognitive function may result from multiple infarcts, leading to so-called multi-infarct dementia, or from intracranial mass lesions, such as subdural hematomas, brain tumors, or granulomas. Central nervous system infections of viral and bacterial origin, or even slow viral disorders such as Creutzfeldt-Jakob disease, are part of the differential diagnosis. Furthermore, metabolic disorders involving vitamin B.sub.12 metabolism, thiamine or folate deficiency, thyroid dysfunction, hepatic and renal failure, as well as drug toxicity may appear as dementia. Nevertheless, when all these secondary causes, many of which are reversible, are eliminated, cerebral atrophy of unknown cause or Alzheimer's disease still covers the largest number of patients.
The definitive diagnosis of Alzheimer's disease is made by pathologic examination of postmortem brain tissue in conjunction with a clinical history of dementia. This diagnosis is based on the presence in brain tissue of intraneuronal neurofibrillary tangles and of neuritic (senile) plaques, which have been correlated with clinical dementia. Although the cause of the abnormal cytoskeletal fibrils remains unknown, neuritic plaques are thought to be composed of degenerating axons and nerve terminals as well as possible astrocytic elements, and they often exhibit a central amyloid protein core. The neurofibrillary tangles are interneuronal aggregates composed of normal and paired helical filaments and presumably consist of several different proteins. The neurohistopathologic identification and counting of neuritic plaques and neurofibrillary tangles requires staining and microscopic examination of several brain sections.
It is problematic, however, that histochemical staining is not always reproducible, neuritic plaques and neurofibrillary tangles are not uniformly distributed, and histopathologic studies are time-consuming and labor-intensive. Moreover, there is no direct evidence that an accumulation of abnormal cytoskeletal fibrils contributes directly to premature dysfunction and death of the neurons; rather, the fibrils may simply be a manifestation of more fundamental cellular changes. The clinical and pathologic progression of Alzheimer's disease is marked by a continuing loss of neurons from the cerebral cortex. However, neuritic plaques and neurofibrillary tangles may occur in nondemented elderly patients, as well as those afflicted with Alzheimer's disease.
Current research to develop both an understanding of the disease and a possible diagnostic test has centered on the amyloid protein and its precursor protein. Theoretically, diagnosis has been based on the deposition of amyloid containing plaques in the cortical region in the brain of individuals affected with Alzheimer's disease. Such diagnostic methods have been disclosed in, for example, U.S. Pat. No. 4,666,829, 4,701,407, 4,816,416 and 4,933,156.
However, amyloid deposits are also found in the brains of aged individuals who have never displayed signs of dementia. For example, a recent article (J. Biol. Chem., 265:15977 (1990)) has shown that there were no differences in the primary structure of precursor amyloid protein from platelets of normal individuals and that of Alzheimer's disease patients. Therefore, while amyloid protein may be involved in Alzheimer's disease, other methods have been pursued to identify characteristics more uniquely related to a patient with Alzheimer's disease. For instance, U.S. Pat. No. 4,727,041, issued to Aroonsakul, discloses a comparative test for the diagnosis of Alzehimer's disease in humans by determining levels of somatotropin and somatomedin-C in the patient's blood sera drawn at intervals following administration of an L-dopa provocative test.
Immunoassay methods have also been developed for detecting the presence of neurochemical markers in Alzheimer's disease patients. U.S. Pat. Nos. 4,728,605 and 4,801,533, issued to Fudenberg et al., disclose comparative methods for diagnosing degenerative disease of the central nervous system, particularly Alzheimer's disease, by measuring immunological parameters and interactive T cells from a patient's peripheral blood. U.S. Pat. No. 4,806,627, issued to Wisniewski et al., discloses protease resistant proteins which comprise scrapie-associated fibrils and a scrapie-specific monoclonal antibody to distinguish certain neurological disease-caused human dementias from Alzheimer's disease.
However, none of the known methods of diagnosis have proven to be a reliable means of detection of Alzheimer's disease in all patients, particularly at early stages of the disease. As a result, alternative methods of diagnosis have been proposed which rely on analyzing the cerebral spinal fluid drawn from the affected patients. For example, Warner in Anal. Chem., 59:1203A-1204A (1987), has proposed a method for detecting Alzheimer's disease related amyloid protein in the cerebral spinal fluid of affected patients.
U.S. Pat. No. 4,874,694, issued to Gandy et al., discloses a diagnostic method for neurological and psychiatric disorders, such as Alzheimer's disease. The method involves incubating cerebrospinal fluid from a patient in the presence of .sup.32 P-labeled adenosine triphosphate (ATP) and a protein kinase which was capable of transferring phosphate from the ATP, followed by electrophoresis. The resulting autoradiographic pattern of the fractionated, labeled sample is then compared with predetermined autoradiographic patterns from known neurological and psychiatric pathologies to ascertain the particular pathology of the patient's cerebrospinal fluid being analyzed. However, the method disadvantageously is based only on autoradiographic patterns. It fails to identify disease-specific marker proteins in the cerebral spinal fluid.
Studies by Khatoon et al. (Ann. of Neurology, 26:210-215 (1989)) have shown that an interaction of an important cellular protein, tubulin, in the formation of microtubules was aberrant in preparations made from the brain tissues of patients with Alzheimer's disease. The inhibition was monitored by measuring the interactions of a radioactive photoaffinity probe of the nucleotide GTP (guanosine-5'-tri-phosphate) with the proteins that require GTP to effect microtubule formation.
Molecules containing azido groups have been shown to form covalent bonds to proteins through reactive nitrene intermediates, generated by low intensity ultraviolet light. Potter & Haley, Meth. in Enzymol., 91:613-633 (1983). In particular, 2- and 8-azido analogues of purine nucleotides have been used as site directed photoprobes to identify nucleotide binding proteins in crude cell extracts. Owens & Haley, J. Biol. Chem. 259:14843-14848 (1984); Atherton et al., Bio. of Reproduction, 32:155-171 (1985). The 2-and 8-azido nucleotides have also been used to map nucleotide binding domains of purified proteins. Khatoon et al., Ann. of Neurology, 26:210-215 (1989); King et al., J. Biol. Chem., 264:10210-10218 (1989); and Dholakia et al., J. Biol. Chem., 264:20638-20642 (1989).
Photoaffinity probes have been used to determine specific nucleotide binding sites on a biologically active recombinant peptide molecule. Campbell et al., PNAS, 87:1243-1246 (1990). The probes have also been used to study enzyme kinetics of purified proteins. Kim et al., J. Biol. Chem., 265:3636-3641 (1990).
Thus, considerable effort has been devoted to developing systems for the definitive diagnosis of Alzheimer's disease in patients. However, until the method of the present invention, no reliable non-invasive test for Alzheimer's disease has been developed. The major drawback of the most definitive determination of Alzheimer's disease known in the art has been that direct analysis of pathological tissue could only be performed postmortem on affected individuals.
Because Alzheimer's disease is progressive in nature, the efficiency of a cure could critically depend upon early detection. Additionally, the value of any new therapy in alleviating or curing the disease could be better ascertained if a rapid, safe and effective diagnostic procedure were available to monitor the progress of Alzheimer's disease patients following treatment.
Therefore, there remains a long-felt need in the art for a reliable, accurate, safe and effective method for the diagnosis of Alzheimer's disease, as well as a means for the differentiation of other neurological diseases and syndromes or psychiatric pathology. A method for the identification and characterization of a disease-specific biochemical marker and the identification of such a marker is needed.